Conditional Loss of Dnmt3a Results in Myeloproliferation and Liver-Specific Myeloid Expansion

Abstract Mutations in the DNA methyltransferase 3A (DNMT3A) gene are frequent in normal karyotype de novo acute myeloid leukemia (AML) (20-35%), chronic myelomonocytic leukemia (CMML) (10-20%) and myelodysplastic syndrome (MDS) (8%). Hematopoietic-specific loss of Dnmt3a in a mouse model leads to acquisition of aberrant self-renewal by the HSCs and expansion of the stem/progenitor compartment in bone marrow transplantation studies. Despite these important insights, the impact of hematopoietic deletion of Dnmt3a on disease phenotype in primary, non-transplanted mice has not been described. Mx1-Cre-mediated Dnmt3a ablation in the hematopoietic system in primary mice led to the development of a myeloproliferative neoplasm (MPN) with a 100% penetrance (n=14) and a median age of onset at 47.7 weeks (survival difference between Dnmt3a KO and control animals p<0.0001, Figure 1A). Loss of Dnmt3a in the hematopoietic compartment resulted in thrombocytopenia (platelet counts 250±251.8 K/μl in Dnmt3a KO vs 1260±292.8 K/μl in controls, p<0.002) and overall anemia (hematocrit 25.25±7.48% vs 44.8±5.83%, p<0.006). Marked expansion of the mature Mac1+Gr1+ myeloid cell population in the peripheral blood was evident by flow cytometric analysis (52.3±18.03% in Dnmt3a knock-outs). Myeloproliferation induced by Dnmt3a loss was characterized by marked, progressive hepatomegaly (liver weights 7.25±1.195 g in Dnmt3a-deleted animals vs 1.61±0.266 g in wild-type controls, p<1.75×10^-8, Figure 1B) with moderate splenomegaly (spleen weights 457.5±379.6 mg vs 79.43±21.19 mg, p<0.033). Histopathological analysis revealed massive myeloid infiltration in spleens and livers leading to complete effacement of organ architecture, left shifted myeloid cells, and occasional blasts. In addition, the presence of megakaryocytes in spleens and livers of Dnmt3a-deleted mice was indicative of extramedullary hematopoiesis. The significant myeloid infiltration of liver parenchyma was confirmed by flow cytometric analysis of liver tissue, with Mac1+Gr1+ myeloid cells making up 66.15±11.93% of all viable cells. In line with previous reports, we observed an increased number of immunophenotypically defined stem (Lin-Sca1+cKit+, LSK, 2.013±1.200% in Dnmt3a-ablated mice vs 0.423±0.052% in controls, a 4.76-fold increase, p<0.014) and granulomonocytic progenitor (GMP, Lin-Sca1-cKit+CD34+FcγR+, 2.713±1.593% vs 1.278±0.451%, a 2.12-fold increase, p<0.024) cells in the bone marrow. Consistent with extramedullary hematopoiesis, we were able to detect expanded LSK cell populations in livers and spleens of Dnmt3a-deleted mice. Notably, the myeloid disease phenotype induced by Dnmt3a loss was fully transplantable, including the marked hepatomegaly; these data demonstrate that the liver-specific expansion reflects a cell-autonomous mechanism. To assess relative tropism for different target organs, we next performed homing studies where Dnmt3a-deleted bone marrow cells were competed against wild-type counterparts in lethally irradiated hosts. 48 hours after transplantation, we observed increased tropism of the Dnmt3aΔ/Δ BM cells to the liver and spleen, whereas control cells preferentially localized to the bone marrow (difference between homing to bone marrow and spleen/liver p<0.0115, Figure 1C). These data demonstrate that altered homing and tissue tropism of Dnmt3a KO hematopoietic cells promote extramedullary hematopoiesis and liver involvement. ERRBS and gene expression profiles by RNA-seq in stem and progenitor cell populations demonstrated differential regulation of key biologic pathways, including self-renewal, hematopoietic lineage commitment and differentiation, and heterotypic cell-cell interactions. In conclusion, our studies show that ablation of Dnmt3a in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous liver tropism and marked extramedullary hematopoiesis. These data demonstrate, in addition to its established role in controlling self-renewal, Dnmt3a serves as an important regulator of the myeloid compartment that limits expansion of myeloid progenitors in vivo. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

Blood ◽

10.1182/blood.v122.21.476.476 ◽

2013 ◽

Vol 122 (21) ◽

pp. 476-476

Author(s):

Alan H. Shih ◽

Yanwen Jiang ◽

Kaitlyn Shank ◽

Suveg Pandey ◽

Agnes Viale ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Targeted Therapies ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Leukemia Stem Cells ◽

Self Renewal ◽

In Vivo Models ◽

Flow Cytometric

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj <0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR<0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

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Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

Blood ◽

10.1182/blood.v112.11.73.73 ◽

2008 ◽

Vol 112 (11) ◽

pp. 73-73

Author(s):

Rebecca L Porter ◽

Benjamin J Frisch ◽

Regis J O’Keefe ◽

Laura M Calvi

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Prostaglandin E2 ◽

Flow Cytometric Analysis ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions ◽

Flow Cytometric ◽

Incorporated Brdu

Abstract HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

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ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽

10.1182/blood.v128.22.1051.1051 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1051-1051

Author(s):

Vikas Madan ◽

Lin Han ◽

Norimichi Hattori ◽

Anand Mayakonda ◽

Qiao-Yang Sun ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Research Funding ◽

Hematopoietic Differentiation ◽

Wild Type ◽

Flow Cytometric ◽

Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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Ap2a2 Is Crucial for LT-HSC Quiescence

Blood ◽

10.1182/blood.v128.22.2642.2642 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2642-2642

Author(s):

Stephen B Ting ◽

Sara Rhost ◽

Sarah Ghotb

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Fold Increase ◽

Wild Type ◽

Lacz Reporter ◽

Adult Survivor ◽

Self Renewal ◽

Post Transplant

Abstract Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150+48-LSK GFPhigh as opposed to the more cycling GFPlow HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150+48-LSK GFPhigh HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150+48-LSK long-term (LT-) versus CD150-48-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150+48-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function. Disclosures No relevant conflicts of interest to declare.

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Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Blood ◽

10.1182/blood.v116.21.404.404 ◽

2010 ◽

Vol 116 (21) ◽

pp. 404-404 ◽

Cited By ~ 1

Author(s):

Heather A Himburg ◽

Pamela Daher ◽

J. Lauren Russell ◽

Phuong Doan ◽

Mamle Quarmyne ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Signaling Pathways ◽

Fold Increase ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Hscs ◽

Necessary And Sufficient

Abstract Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

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The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.4694.4694 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4694-4694

Author(s):

Hong Xu ◽

Ziqiang Zhu ◽

Yiming Huang ◽

Suzanne T. Ildstad

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Dose Titration ◽

Great Promise ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

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Intermittent in Vivo Parathyroid Hormone (PTH) Treatment Results in Jagged 1 (Jag 1) Dependent and Independent Effects On Hematopoietic Stem Cells (HSCs)

Blood ◽

10.1182/blood.v120.21.642.642 ◽

2012 ◽

Vol 120 (21) ◽

pp. 642-642

Author(s):

Benjamin J Frisch ◽

Ismat Shafiq ◽

Rebecca L Porter ◽

Jonathan M Weber ◽

Julianne N Smith ◽

...

Keyword(s):

Caspase 3 ◽

Flow Cytometric Analysis ◽

Osteoblastic Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Hsc Niche ◽

Independent Mechanism ◽

Independent Effects

Abstract Abstract 642 HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures: No relevant conflicts of interest to declare.

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Agonistic Anti-DR3 Antibody Expands Treg and Reduces Experimental Acute Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v122.21.135.135 ◽

2013 ◽

Vol 122 (21) ◽

pp. 135-135

Author(s):

Byung-Su Kim ◽

Antonio Pierini ◽

Dominik Schneidawind ◽

Jeanette Baker ◽

Hidekazu Nishikii ◽

...

Keyword(s):

T Cell ◽

Lymph Nodes ◽

Acute Gvhd ◽

Ex Vivo ◽

Flow Cytometric Analysis ◽

Wild Type ◽

Graft Versus Host ◽

Flow Cytometric ◽

Acute Graft

Abstract Regulatory T cells with the phenotype of CD4+CD25+FoxP3+ (Treg) decrease acute graft-versus-host disease (GVHD) while preserving graft-versus-leukemia effects. However, their low frequency limits clinical translation. One approach has been to expand Treg ex vivo using cytokines (IL-2/TGFb) and antibodies (anti-CD3/anti-CD28). Another approach may be to develop strategies to expand Treg in vivo. Death Receptor 3 (DR3) is a member of the TNF superfamily and its agonistic antibody (4C12) was reported to preferentially activate Treg. We investigated the role of DR3 stimulation in expanding Treg and decreasing acute GVHD in a murine model. Two approaches were investigated. The first approach was to utilize 4C12 to treat B6 donor mice (CD45.1/luc+) and isolate conventional T cells (Tcon) four days later from lymph nodes and spleen using anti-CD4 and anti-CD8 beads. Tcon from 4C12-treated donors were consistently enriched with an increased percentage and absolute numbers of Foxp3+CD4+ cells compared to Tcon from isotype-treated donors (25.6% vs. 12.6%, p<0.01). In the mixed lymphocyte reaction Tcon from 4C12-treated donors were less proliferative compared to Tcon from isotype-treated donors. Moreover, CD4+CD25+ cells from 4C12-treated donors showed stronger suppressive effects suggesting that not only were Treg expanded but that they were also activated and more functional. Tcon (1x10^6) from 4C12 vs isotype treated luc+ C57Bl/6 donors were injected into lethally irradiated Balb/c recipients along with 5x10^6 T cell-depleted BM cells from wild-type B6 mice. Animals that received 4C12 treated Tcon had significantly lower GVHD-related score and better survival than Balb/c mice that received Tcon from isotype-treated donors (n=10 for each group, p<0.01). Bioluminescence imaging (BLI) showed recipient mice that received Tcon from 4C12-treated donors had decreased Tcon expansion early after transplantation compared to recipients of Tcon from isotype-treated donors (Day 3, p=0.04; Day 6, p=0.04). Flow cytometric analysis of lymph nodes and spleens harvested from recipients of Tcon from 4C12-treated donors confirmed that significantly higher Treg levels were maintained in donor Tcon on Day 4 and Day 7 after transplantation. In other experiments, recipients of Tcon from 4C12-treated donors still had significantly higher Treg levels in donor Tcon even 20 days after infusion. The second approach was to treat lethally irradiated recipient Balb/c mice with 4C12 or isotype antibody intraperitoneally one hour after the injection of 5x10^6 T cell-depleted BM cells from wild-type B6 mice. Tcon (1x10^6) from CD45.1 luc+ B6 mice were injected on Day 2. Recipient mice treated with 4C12 had significantly lower GVHD-related score and better survival than isotype-treated recipients (n=5 for each group, p<0.01). BLI also showed 4C12-treated mice had decreased T cell expansion (Day 8, p=0.03; Day 20, p<0.01). Lymph nodes and spleens were harvested on Day 5 after in vivo BrdU labeling for flow cytometric analysis. The host CD4 T cell population from 4C12-treated recipients had more Foxp3+ cells that also incorporated significantly more BrdU. In summary, stimulation of Treg through DR3 resulted in enhanced number and function of Treg which was associated with less GVHD by either treating the donor mice or recipients at the time of transplant. This approach of Treg stimulation could serve as an alternative approach to ex vivo Treg expansion to enhance Treg function resulting in less morbidity and better survival with reduced GVHD risk. Together our data suggest that agonistic anti-DR3 antibody stimulation can effectively activate and expand Treg resulting in decreased acute GVHD in a murine GVHD model. Disclosures: No relevant conflicts of interest to declare.

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Osteal Macrophages and Megakaryocytes Increase Adipogenesis

Proceedings of IMPRS ◽

10.18060/25739 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Nikhil Tewari ◽

Deepa Kanagasabapathy ◽

Rachel J. Blosser ◽

Edward F. Srour ◽

Angela Bruzzaniti ◽

...

Keyword(s):

Bone Marrow ◽

Cell Sorting ◽

Fetal Liver ◽

Fold Increase ◽

Wild Type ◽

Bone Marrow Adipose Tissue ◽

New Treatments

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified. We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

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DNMT3A with Leukemia-Associated R882 Mutations Promotes Fitness Advantage through Functional Heterogeneity within HSCs

Blood ◽

10.1182/blood-2021-151007 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2151-2151

Author(s):

Zachary Zaroogian ◽

Chamara Gunaratne ◽

Daniil Shabashvili ◽

Cassandra Berntsen ◽

Yang Feng ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Function ◽

Clonal Evolution ◽

Single Amino Acid ◽

Wild Type ◽

Self Renewal ◽

Clonal Hematopoiesis ◽

Test Cells

Abstract Mutations in DNA damage regulators and epigenetic modifier genes including those in DNA methyltransferase 3A (DNMT3A) are common in pre-malignant clonal hematopoiesis (CH) and are recurrent in myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While loss-of-function (LOF) mutations in DNMT3A are predominant in CH, single amino-acid substitutions at residue 882, usually from an arginine to a histidine or cysteine (DNMT3A R882H / DNMT3A R882C), are strongly enriched in AML, suggesting a role in promoting pre-malignant clonal evolution. In this study, we compared and contrasted the effects of DNMT3A R882 and LOF on hematopoietic stem cell (HSC) function by investigating self-renewal capacity in vitro and bone marrow repopulation potential in vivo in a genetic mouse model. Bone marrow (BM) from mice conditionally expressing Dnmt3a R878H (equivalent to human DNMT3A R882H) from the endogenous locus, denoted as +/mut, was subjected to colony forming unit (CFU) assays in semisolid media (MethoCult GF M3434, Stem Cell Technologies) to detect hematopoietic progenitors with serial replating every 14 days as a surrogate measure of stem cell self-renewal. Dnmt3a +/mut cells were compared to bone marrow derived from mice with heterozygous or homozygous Dnmt3a knockout (+/- and -/-, respectively), or wild-type control (+/+). Dnmt3a +/- BM cells showed a trend towards a slight increase in the number of replatings compared to wild-type controls (2.83±0.41 in +/+ vs 3.75±0.89 in +/-, n=6,8, Mann-Whitney test p=0.053). In contrast, Dnmt3a +/mut BM occasionally resulted in a significant gain of self-renewal with replating for 6+ passages observed in 4/10 independent trials (6.00±2.06 in +/mut, n=10, p=0.001 vs +/+), yet failed to reach continuous replating of complete Dnmt3a loss that served as a positive control (terminated at passage 10, n=5). Substantial heterogeneity in the Dnmt3a +/mut self-renewal capacity ex vivo was further reflected by increased statistical variance (Welch's F-test p=0.037 compared to +/-). To extend these studies to the in vivo setting, we performed gold-standard serial competitive bone marrow repopulation assays. Here, 250 FACS-sorted Lineage -Sca1 +cKit +CD48 -CD150 + (LSK-SLAM) test cells marked with CD45.2 were mixed with 0.25x10 6 unfractionated wild-type competitor bone marrow cells marked with CD45.1 and engrafted into pre-conditioned congenic recipients (CD45.1). Peripheral blood CD45.2/CD45.1 chimerism is used as a readout for the ability of test cells to reconstitute hematopoiesis; self-renewal potential is assessed via serial retransplantation. In this setting, Dnmt3a +/+ cells showed a decline in their ability to reconstitute hematopoiesis in secondary transplant recipients, while Dnmt3a haploinsufficiency resulted in a competitive advantage over wild-type (CD45.2 chimerism 2.4±2.7% in +/+ vs 75.6±13.0% in +/-, n=4, Welch's t-test p=0.001). In contrast, Dnmt3a +/mut cells displayed a high degree of variability in their long-term stem cell function in some cases yielding high levels of chimerism (CD45.2 >40% in 3/9 recipients) and in other cases not (26.0±21.0%, n=9, p=0.01 vs +/+), in agreement with CFU assays. Collectively, these data suggest that the DNMT3A R882 mutations endow normal HSCs with phenotypic heterogeneity yielding a population of cells with variable stem cell function, some of which may have higher fitness thus accelerating clonal evolution towards malignancy. These findings will be built upon in future experiments aiming to identify underlying molecular mechanisms and to improve our understanding of the pathophysiology and prognostication of clonal hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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